Differentiation of mesenchymal stem cells in heparin-containing hydrogels via coculture with osteoblasts

Abstract

The therapeutic potency of delivered mesenchymal stem cells (MSCs) in tissue engineering applications may be improved by priming cells toward a differentiated state via coculture with native, differentiated cells prior to implantation; however, there is a lack of understanding in what may be the most efficacious method. The objective of this study was to investigate the role of negatively-charged heparin in priming hydrogel-encapsulated MSCs toward the osteoblastic lineage during coculture with a monolayer of osteoblasts in the absence of dexamethasone. MSCs encapsulated with higher amounts of heparin and cocultured with osteoblasts exhibited an over 36-fold increase in alkaline phosphatase activity and 13-fold increase in calcium accumulation by day 21, compared to MSCs cocultured with MSCs at the same heparin content. Moreover, hydrogels with higher amounts of heparin and cocultured with osteoblasts exhibited enhanced mineralization on the edges, suggesting that heparin may be important in sequestering osteoblast-secreted soluble factors, particularly on the surfaces of hydrogels. The ability of heparin to selectively interact with soluble positively-charged proteins from the surroundings was confirmed through protein labeling and microscopy. These results suggest that heparin-containing hydrogels as part of a coculture system can be utilized as a versatile platform to study and enhance priming of MSCs toward various cell types for a wide variety of regenerative medicine-based therapies.

Hydrolysis and sulfation pattern effects on release of bioactive bone morphogenetic protein-2 from heparin-based microparticles

“…For 100% heparin MPs, the high density of heparin binding sites may sterically hinder some sites from being occupied, whereas within 10% and 1% heparin MPs, the increased distance between heparin binding sites could potentially allow for more efficient BMP-2 binding. Previous studies in our laboratory found that positively charged protein penetration into heparin-based hydrogels decreased with increasing heparin content, again supporting the idea that MPs with less heparin could load growth factors more efficiently than 100% heparin MPs 16 . Taken together, these results suggest that reduced heparin content within MPs is still adequate for efficient BMP-2 loading.…”

“…However, we and other laboratories 12–16 are exploring the use of glycosaminoglycans (GAGs) to potentially reduce burst release of BMP-2 and prolong growth factor bioactivity 9,11,17 . GAGs are linear polysaccharides found within the extracellular matrix (ECM) either as free chains or, more often, covalently bound to a polypeptide core, collectively known as proteoglycans 18,19 .…”

Heparin microparticle effects on presentation and bioactivity of bone morphogenetic protein-2

“…Heparin ammonium salt from porcine intestinal mucosa (17–19 kDa; Sigma-Aldrich, St. Louis, MO) was conjugated with N-(3-Aminopropyl)methacrylamide (APMAm; Polysciences, Warrington, PA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Thermoscientific, Rockford, IL) and N-hydroxysulfosuccinimide (Sulfo-NHS; Thermoscientific, Rockford, IL) as described in previous protocols [44, 45] (Figure 1A). EDC/Sulfo-NHS chemistry causes activation of the carboxyl groups on heparin and subsequent methacrylamide substitution via covalent bonds created with the primary amines on APMAm.…”

Heparin-based hydrogels with tunable sulfation & degradation for anti-inflammatory small molecule delivery

“…12,33-39 Positively-charged proteins such as stromal cell-derived factor-1 (SDF-1), bone morphogenetic protein-2 (BMP-2), and VEGF were then loaded on or into these biomaterials for controlled release based on electrostatic affinity. 12,33-39 …”